The importance of PUFAs is undisputed. For example, certain PUFAs are important biological components of healthy cells and are recognized as: “essential” fatty acids that cannot be synthesized de novo in mammals and instead must be obtained either in the diet or derived by further elongation and desaturation of linoleic acid (LA; 18:2 omega-6) or α-linolenic acid (ALA; 18:3 omega-3); constituents of plasma membranes of cells, where they may be found in such forms as phospholipids or triacylglycerols; necessary for proper development (particularly in the developing infant brain) and for tissue formation and repair; and, precursors to several biologically active eicosanoids of importance in mammals (e.g., prostacyclins, eicosanoids, leukotrienes, prostaglandins). Additionally, a high intake of long-chain omega-3 PUFAs produces cardiovascular protective effects (Dyerberg et al., Amer. J. Clin. Nutr. 28:958-966 (1975); Dyerberg et al., Lancet 2(8081):117-119 (1978); Shimokawa, H., World Rev. Nutr. Diet 88:100-108 (2001); von Schacky et al., World Rev. Nutr. Diet 88:90-99 (2001)). Numerous other studies document wide-ranging health benefits conferred by administration of omega-3 and/or omega-6 PUFAs against a variety of symptoms and diseases (e.g., asthma, psoriasis, eczema, diabetes, cancer).
Today, a variety of different hosts including plants, algae, fungi, and yeast are being investigated as means for commercial PUFA production via numerous divergent efforts. Although the natural PUFA-producing abilities of the host organisms are sometimes essential to a given methodology, genetic engineering has also proven that the natural abilities of some hosts (even those natively limited to LA and ALA fatty acid production) can be substantially altered to result in high-level production of various long-chain omega-3/omega-6 PUFAs. Whether this effect is the result of natural abilities or recombinant technology, arachidonic acid (ARA; 20:4 omega-6), eicosapentaenoic acid (EPA; 20:5 omega-3), and docosahexaenoic acid (DHA; 22:6 omega-3) all require expression of either the delta-9 elongase/delta-8 desaturase pathway (which operates in some organisms, such as euglenoid species and which is characterized by the production of eicosadienoic acid (EDA; 20:2 omega-6) and/or eicosatrienoic acid (ETrA; 20:3 omega-3)) or the delta-6 desaturase/delta-6 elongase pathway (which is predominantly found in algae, mosses, fungi, nematodes and humans and which is characterized by the production of gamma-linolenic acid (GLA; 18:3 omega-6) and/or stearidonic acid (STA; 18:4 omega-3)) (FIG. 1). A delta-6 elongase is also known as a C18/20 elongase.
The delta-8 desaturase enzymes identified thus far have the ability to convert both EDA to dihomo gamma-linolenic acid (DGLA (also known as HGLA); 20:3, n-6) and ETrA to eicosatetraenoic acid (ETA; 20:4, n-3). ARA and EPA are subsequently synthesized from DGLA and ETA, respectively, following reaction with a delta-5 desaturase. DHA synthesis, however, requires the subsequent expression of an additional C20/22 elongase and a delta-4 desaturase. Most C20/22 elongases identified so far have the primary ability to convert EPA to DPA, with secondary activity in converting arachidonic acid (ARA; 20:4 omega-6) to docosatetraenoic acid (DTA; 22:4 omega-6), while most delta-4 desaturase enzymes identified so far have the primary ability to convert DPA to DHA, with secondary activity in converting docosatetraenoic acid (DTA; 22:4 omega-6) to ω-6 docosapentaenoic acid (DPAn-6; 22:5 omega-6).
Based on the role C20/22 elongase and delta-4 desaturase enzymes play in the synthesis of DHA, there has been considerable effort to identify and characterize these enzymes from various sources. As such, numerous C20/22 elongases have been disclosed in both the open literature and the patent literature (e.g., Pavlova sp. CCMP459 (GenBank Accession No. AAV33630), Ostreococcus tauri (GenBank Accession No. AAV67798) and Thalassiosira pseudonana (GenBank Accession No. AAV67800)). Similarly, the following delta-4 desaturases have been disclosed: Euglena gracilis (SEQ ID NO:13; GenBank Accession No. AAQ19605; Meyer et al., Biochemistry, 42(32):9779-9788 (2003)); Thalassiosira pseudonana (SEQ ID NO:29; GenBank Accession No. AAX14506; Tonon et al., FEBS J., 272(13):3401-3412 (2005)); Thraustochytrium aureum (SEQ ID NO:27; GenBank Accession No. AAN75707); Thraustochytrium sp. (GenBank Accession No. CAD42496; U.S. Pat. No. 7,087,432); Schizochytrium aggregatum (SEQ ID NO:28; PCT Publication No. WO 2002/090493); Pavlova lutheri (GenBank Accession No. AAQ98793); and Isochrysis galbana (SEQ ID NO:30; GenBank Accession No. AAV33631; Pereira et al., Biochem. J., 384(2):357-366 (2004); PCT Publication No. WO 2002/090493)].
Applicants' Assignee has a number of patent applications concerning the production of PUFAs in oleaginous yeasts (i.e., Yarrowia lipolytica), including, for example: U.S. Pat. No. 7,238,482 and No. 7,125,672; U.S. application Ser. No. 11/265,761 (filed Nov. 2, 2005); U.S. application Ser. No. 11/264,784 (filed Nov. 1, 2005); U.S. application Ser. No. 11/264,737 (filed Nov. 1, 2005).
Relatedly, PCT Publication No. WO 2004/071467 (published Aug. 26, 2004) concerns the production of PUFAs in plants, while PCT Publication No. WO 2004/071178 (published Aug. 26, 2004) concerns annexin promoters and their use in expression of transgenes in plants. Both are Applicants' Assignee's copending applications.